Rapid and simple tests that can be used to accurately diagnose the occurrence of MIs are extremely important. One biochemical test that can be used in the diagnosis of MI is the MB isoenzyme of creatine kinase ("CK-MB"). However, CK-MB can also be found in skeletal muscle and in blood after skeletal muscle injury. Thus, CK-MB is not completely specific for cardiac muscle. See, e.g., Cummins, et al. (1987), "Cardiac-Specific Troponin I Radioimmunoassay in the Diagnosis of Acute Myocardial Infarction", American Heart Journal, Jun. 1987, Vol. 113, No. 6. Another disadvantage of the CK-MB test is that the amount of CK-MB in the skeletal muscle varies with the degree of skeletal muscle regeneration, information which may often not be known when administering a test or analyzing a test result for MI. Another disadvantage of the CK-MB test is that CK-MB remains elevated for only 2-3 days after the onset of chest pain. For patients admitted after that time, the CK-MB test will be of limited, if any, value. See, e.g., Cummins, et al. (1987). Thus, due to the lack of specificity of the CK-MB test, and the limited time frame for its use as a diagnostic tool, CK-MB is not the MI test of choice.
Other enzyme assays exist, such as lactate dehydrogenase (LDH) and glutamic oxaloacetic transaminase (GOT). However, the frequent serial measurements required in the very early hours after the onset of chest pain can present difficulties for an absolute specific diagnosis. See, e.g., Larue, et al. (1992), "New Monoclonal Antibodies as Probes for Human Cardiac Troponin I: Epitopic Analysis With Synthetic Peptides", Molecular Immunology, Vol. 29, No. 2, pp. 271-278. Thus, the prior art has recognized the need for an accurate cardiac-specific biological parameter detectable in serum very soon after MI and remaining present for more than 2-3 days after the onset of MI.
Troponin I (TnI) is the inhibitory sub-unit of Troponin, a thin filament regulatory protein complex, which confers calcium sensitivity to the cardiac and striated muscle. The Troponin complex consists of three subunits: Troponin T (TnT), the tropomyosin binding subunit, Troponin C (TnC), the Ca++ binding subunit; and TnI, which inhibits the actomyosin Mg++ ATPase.
Troponin I exists in three isoforms: two skeletal muscle isoforms (fast and slow) (Molecular Weight=19,800 daltons) and a cardiac TnI isoform (cTnI) with an additional 31 residues (human TnI) on the N-terminus resulting in a molecular weight of 23,000 daltons. Cardiac TnI is uniquely located in the myocardium where it is the only isotype (Cummins, P. and Perry, V. S., (1978) Biochem. J. 171:251-259. The amino acid sequence of cardiac TnI from various species has been determined. There is little difference in the primary structure between human cardiac TnI (209 amino acids) and bovine cardiac TnI (211 amino acids). More differences are seen between cardiac TnI and skeletal muscle TnI. (Leszky et al. (1988) Biochemistry, Vol. 27, pp. 2821-2827).
Cardiac TnI (cTnI) rapidly appears in human serum (within approximately 4 to 6 hours) following a MI. It reaches a peak level after 18-24 hours and remains at elevated levels in the blood stream for up to 6 to 7 days. Thus, immunoassays which can test for human cTnI are valuable to the medical community and to the public.
The TnI released into circulation is very specific for myocardial injury. Little information is presently known about Troponin I present in the circulation after being released from the heart. Knowledge about TnI in MI patient serum is very important for studying the effects of MI on heart muscle as well as for developing assays to assist in the diagnosis and treatment of MI patients.
It is desirable to use an immunologically reactive human cTnI form comparable to that detected in MI patient serum. We found that MI patient serum contains TnI fragment(s) which is the result of the C-terminal processing of cTnI molecule. The high sequence homology found in the C-terminal region between cardiac TnI and skeletal muscle TnI (Larue et al. (1992); Vallins et al. (1990) FEBS Lett. Vol. 270, pp. 57-61; Leszky et al. (1988)) produce TnI antibodies directed against this region having non-cardiac specificity. (Larue et al. (1992)). Our data and Larue et al. 1992 suggest that most of the known cTnI specific antibodies have their epitopes located approximately in the first 75% of the TnI molecule. Therefore, this portion of the TnI molecule should function as a MI specific cTnI isoform in most immunoassay systems. Consequently, MI assays that look at cTnI have become important.
However, present TnI immunoassays use different TnI antigens in the calibrators and the controls. For example, Dade International Inc. (hereinafter sometimes referred to as "Dade") presently sells a cTnI Immunoassay Kit in Europe and U.S.A. using a synthetic peptide in the calibrators and in the controls. This product is the STRATUS.RTM. Cardiac Troponin-I assay, a radial partition immunoassay. It would be advantageous to use a native human TnI form in the calibrators and controls in order to more accurately simulate the conditions in patient serum.
The use of the native human peptide is not practical because native human cTnI form is difficult to obtain due to the scarcity of human heart muscle. Moreover, native human cTnI is highly subject to proteolytic degradation during purification. The availability of human recombinant TnI ("r-TnI") can facilitate the production of this cTnI form. The r-TnI, unlike the native human cTnI, can be produced and purified in acceptable quantities. As expressed by Dade, the primary structure of r-TnI contains 226 amino acids (SEQ ID NO: 1); 209 of them represent the TnI sequence (SEQ ID NO: 2). In addition to the primary sequence of cTnI (SEQ ID NO: 2), r-TnI has a leading sequence of 8 amino acids (MASMTLWM) on the N-terminal, and a tail sequence of 9 amino acids (PMVHHHHHH) on the C-terminal (SEQ ID NO: 1). The primary structure of the r-TnI molecule has methionine residues at positions -7, -4, 0, 153, 154, 200 and 211 (SEQ ID NO: 1).
U.S. application Ser. No. 08/564,526 discloses a human cTnI fragment generated from human r-TnI by chemical cleavage. The cleavage of r-TnI by cyanogen bromide (CNBr) results in a major polypeptide of 153 amino acids, hereinafter referred to as the "CNBr-cTnI isoform" (SEQ ID NO: 3). The CNBr-cTnI isoform represents 73% of the primary structure of human cTnI and is immunologically more reactive than r-TnI. The purified CNBr-cTnI isoform has an average of 3-4 times more reactivity than r-TnI and lower non-specific binding, as measured by radial partition immunoassay. The molecular size of the CNBr-cTnI isoform is comparable in molecular weight to a major degradation product of native cardiac TnI in MI patient serum. The CNBr-cTnI isoform can be used as calibrators or controls in various cTnI immunoassays. The CNBr-cTnI isoform has increased stability over the synthetic peptide currently used in the Dade TnI immunoassay.
Identifying stable and active cTnI forms that can be used as calibrators, controls or in other areas would be very important. Even though the 153 amino acid CNBr-isoform of r-TnI has a greater immunoreactivity than r-TnI and is more stable that the synthetic peptide currently used, there are advantages to using the native TnI as it exists in MI patient serum. For example, as mentioned above, the use of native cTnI in controls and calibrators in assays to test for cTnI would more accurately simulate the conditions in the test sample, leading to more accurate assay results.
Furthermore, while the assays presently used to detect cTnI in MI patient serum are sandwich assays, other types of assays are possible. For example, competitive assays can be used to detect cTnI. It would be useful to use an immunologically reactive human cardiac TnI form comparable to that in MI patient serum in a competitive-type assay for cTnI.